1. Field of the Invention
The present invention relates to a process for increasing the free pool lysine content in maize through selection in tissue culture.
2. Description of the Prior Art
Cereal grains are a major source of vegetable protein. Maize (Zea mays L.) contributes approximately one-fourth of the total cereal protein produced. However, maize has a low content of certain essential amino acids, especially lysine, methionine and typtophan. As a result, maize in the diet must be supplemented with food containing these amino acids in order to provide a balanced diet. One goal of plant breeding in maize has been to increase the amount of lysine and tryptophan present in the seed. At least two approaches can be visualized to accomplish an increase in these amino acids. The first approach is to increase the lysine or tryptophan content in the proteins found in the maize kernel. The second is to increase the free pool (endogenous) lysine or tryptophan content within the maize kernel.
The first significant breeding results with regard to changes of the protein composition of maize kernels in the direction desired was the discovery by Mertz, E. J., et al. (Science 145, 279 (1964)) that the protein composition of maize endosperm could be drastically changed by a single gene (opaque-2). In the following year, the same authors reported a second mutant gene (floury-2) which changed the protein composition of maize endosperm in a similar way (Nelson, O. E., et al., Science 150, 1469 (1965)). It was found that, besides similar changes in lysine and tryptophan, floury-2 also has a higher methionine content.
It has been demonstrated (Nelson, O. E., Genet.Agron. 21, 209 (1967)) that the different amino acid composition of the two maize mutants is chiefly due to the modification of the relative amounts of protein fractions, i.e., a partial suppression of the prolamine and its replacement by other fractions rich in lysine and tryptophan.
Considering the agronomic performance and especially the yield, it seems that opaque-2 is somewhat inferior to normal maize, chiefly due to its lighter kernels. In populations where opaque-2 had been introduced into inbred lines, it has been found that the percentage weight loss of opaque-2 kernels as compared to normal sibs varied from less than 5% to 40%, depending on the inbred line.
Although opaque-2 stocks produce in general smaller and lighter kernels than normal stocks, it has been pointed out that it would be unsafe to conclude that opaque-2 types have necessarily a lower yield than normal isogenic types. The data indicate that modifier genes affect kernel size in opaque-2 homozygotes; therefore appropriate selection in segregating populations should be effective in improving that trait. Other researchers are of a similar opinion and point out that the density of opaque-2 kernels is dependent on the genetic background which would make it possible to select lines in which opaque-2 shows higher densities.
The opaque-2 gene has been incorporated into corn hybrids which commonly had a lower yield than their normal hybrid counterparts. Recently, however, improvements in yield have spurred renewed interest in high lysine corn usage.
Although much effort has been expended to increase the lysine content of maize proteins, very little effort has gone towards the increase of lysine content in maize by increasing the free pool lysine content.
Plant regeneration from cells in culture is essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation or in vitro selection, and for the use of genetic engineering in producing new varieties. Although plants can be regenerated from tissue culture of several varieties of corn, there are many varieties for which this has not been accomplished using similar techniques.
In recent years, plant cell culture successes have had a considerable influence on the understanding of the respective roles of cell and organism in control of plant growth and development. Isolated plant cells have been shown to be amenable to in vitro culture and complete plants have been regenerated from cultures derived from somatic tissues, either directly via somatic embryogenesis or indirectly via organogenesis. Generally, the regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, especially growth regulators. Early investigations of certain plant species have suggested that exogenous auxin concentration is a major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of embryoid formation. In other species, exposure to a definite balance of auxin and cytokinin leads to the occurrence of organogenesis (shoots, then roots). Although several genotypes of corn have been regenerated using these techniques, no process is generally applicable to most genotypes of corn. Many genotypes remain extremely difficult, if not impossible, to culture using the prior processes.
The process which has become the standard system for corn tissue culture is described by Green et al., Crop Science 15, 417 (1975). In this process, immature embryos were plated onto a callus induction medium which comprises the MS mineral salts, Straus vitamins and amino acids (glycine, asparagine, niacin, thiamine, pyridoxine and pantothenic acid), 2% sucrose, 0.8% agar and a hormone selected from 2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid (p-CPA), .alpha.-naphthaleneacetic acid (NAA), 2-isopentyladenine (2-ip) or mixtures thereof. Hormone concentrations which were useful were 2 mg/l 2,4-D and a mixture of 1 mg/l 2,4-D, 4 mg/l NAA and 0.05 mg/l 2-ip. Plantlets were regenerated by subculturing the callus on medium containing reduced hormone concentrations. Regeneration was then accomplished on medium containing 0.25 mg/l 2,4-D or a mixture of 1 mg/l NAA and 0.05 mg/l 2-ip, respectively. All culturing was conducted in a 16 hour light/8 hour dark cycle for 3-4 week intervals before transfer. This reference reports that callus induction did not occur in one of five genotypes tested.
Similar results with different media have been demonstrated by Freeling et al., Maydica 21, 97 (1976); Vasil et al., Theor.Appl. Genet. 66, 285 (1983); Edallo et al., Maydica 26, 39 (1981); Lu et al., Theor.Appl.Genet. 62, 109 (1982); Gegenbach et al., Proc.Nat. Acad.Sci.USA 74, 5113 (1977); and Green et al., Crop Science 14, 54 (1974). The latter reference also demonstrates genotype effects on callus induction.
Although this procedure has generally been unsuccessful for regenerating plants from all maize genotypes, the regeneration of most genotypes is now possible through the substitution of dicamba for 2,4-D in the media. See published European Application No. 0 177 738 and Duncan et al., Planta 165, 322 (1985).